The role of tyrosine residues of protein L in the binding reaction with human IgG

2001 ◽  
Vol 29 (1) ◽  
pp. A22-A22
Author(s):  
N. G. Housden ◽  
J. A. Beckingham ◽  
N. M. Muir ◽  
S. P. Bottomley ◽  
M. G. Gore
Keyword(s):  
Protein L ◽  
Human Igg ◽  
Binding Reaction ◽  
Tyrosine Residues

Studies on a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus: the role of tyrosine-53 in the reaction with human IgG

2001 ◽  
Vol 353 (2) ◽  
pp. 395-401 ◽  
Author(s):  
Jennifer A. BECKINGHAM ◽  
Nicholas G. HOUSDEN ◽  
Nicola M. MUIR ◽  
Stephen P. BOTTOMLEY ◽  
Michael G. GORE
Keyword(s):  
Chain Complex ◽  
Binding Domain ◽  
Protein L ◽  
Human Igg ◽  
Tyrosine Residues ◽  
Kd Values ◽  
Trp Mutant ◽  
The Stability ◽  
Immunoglobulin Binding

Chemical modification experiments with tetranitromethane (TNM) have been used to investigate the role of tyrosine residues in the formation of the complex between PpL (the single Ig-binding domain of protein L, isolated from P. magnus strain 3316) and the kappa light chain (κ-chain). Reaction of PpL with TNM causes the modification of 1.9 equiv. of tyrosine (Tyr51 and Tyr53) and results in an approx. 140-fold decrease in affinity for human IgG. Similar experiments with mutated PpL proteins suggest that nitration predominantly inactivates the protein by modification of Tyr53. Reduction of the nitrotyrosine groups to aminotyrosine by incubation with sodium hydrosulphite does not restore high affinity for IgG. Modification of κ-chain by TNM resulted in the nitration of 3.1±0.09 tyrosine residues. When the PpLŐκ-chain complex was incubated with TNM, 4.1±0.04 tyrosine residues were nitrated, indicating that one tyrosine residue previously modified by the reagent was protected from TNM when the proteins are in complex with each other. The Kd for the equilibrium between PpL, human IgG and their complex has been shown by ELISA to be 112±20nM. A similar value (153±33nM) was obtained for the complex formed between IgG and the Tyr64 → Trp mutant (Y64W). However, the Kd values for the equilibria involving the PpL mutants Y53F and Y53F,Y64W were found to be 3.2±0.2 and 4.6±1µM respectively. These suggest that the phenol group of Tyr53 in PpL is important to the stability of the PpLŐκ-chain complex.

scholarly journals Comparison of the role of tyrosine residues in human IgG and rabbit IgG in binding of complement subcomponent C1q

1989 ◽  
Vol 257 (3) ◽  
pp. 845-851 ◽  
Author(s):  
M N McCall ◽  
S B Easterbrook-Smith
Keyword(s):  
Receptor Site ◽  
Binding Activity ◽  
Human Igg ◽  
Tryptic Peptides ◽  
Tyrosine Residues ◽  
Rabbit Igg

Treatment of covalently cross-linked or heat-aggregated oligomers of human IgG with 4 mM-tetranitromethane abrogated their C1q-binding activity. In contrast, tetranitromethane modification of rabbit IgG oligomers, under identical conditions, had no effect upon their C1q-binding activity. The tetranitromethane treatment led to nitration of about ten tyrosine residues per IgG molecule in both species, and the modification was specific for tyrosine residues. Reduction of the nitrated protein with Na2S2O4 did not lead to recovery of C1q-binding activity in human IgG oligomers or to loss of activity in rabbit IgG oligomers. Tryptic peptides from the nitrated proteins were isolated and a peptide containing nitrotyrosine-319 was recovered from human IgG, as well as peptides from both species corresponding to the region around nitrotyrosine-278. These data are consistent with the inactivation of C1q-binding activity in human IgG being the result of nitration of tyrosine-319; the rabbit IgG is unaffected by nitration because position 319 is phenylalanine. The evidence supports the C1q-receptor site proposed by Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek [(1980) Nature (London) 288, 338-344]: residues 316-338.

scholarly journals Studies on a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus: the role of tyrosine-53 in the reaction with human IgG

2001 ◽  
Vol 353 (2) ◽  
pp. 395 ◽  
Author(s):  
Jennifer A. BECKINGHAM ◽  
Nicholas G. HOUSDEN ◽  
Nicola M. MUIR ◽  
Stephen P. BOTTOMLEY ◽  
Michael G. GORE
Keyword(s):  
Binding Domain ◽  
Protein L ◽  
Human Igg ◽  
Peptostreptococcus Magnus ◽  
Immunoglobulin Binding

Role of two adjacent cytoplasmic tyrosine residues in MRP1 (ABCC1) transport activity and sensitivity to sulfonylureas

2005 ◽  
Vol 69 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Gwenaëlle Conseil ◽  
Roger G. Deeley ◽  
Susan P.C. Cole
Keyword(s):  
Transport Activity ◽  
Tyrosine Residues

scholarly journals High doses of immunoglobulin G attenuate immune aggregate-mediated complement activation by enhancing physiologic cleavage of C3b in C3bn- IgG complexes

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 184-193 ◽  
Author(s):  
HU Lutz ◽  
P Stammler ◽  
E Jelezarova ◽  
M Nater ◽  
PJ Spath
Keyword(s):  
Inflammatory Diseases ◽  
Factor H ◽  
Human Igg ◽  
Partial Protection ◽  
Beneficial Effects ◽  
High Concentrations ◽  
High Doses ◽  
Immune Aggregates

Abstract Intravenously applied human IgG has beneficial effects in treating inflammatory diseases, presumably because it has a complement attenuating role. This role of IgG was studied in vitro by following C3 activation and inactivation in sera that were supplemented with exogenous human IgG and incubated with immune aggregates. IgG added at 2 to 10 mg/mL stimulated the physiologic inactivation of C3b-containing complexes twofold to threefold in 20% sera. This, in turn, lowered the overall C3 activation by 28%, as new C3 convertases primarily assembled on C3b-containing complexes. Exogenous IgG (5 mg/mL) also stimulated inactivation of purified C3b2-IgG complexes, whereby their half-life dropped from 3–4 to 1.5 minutes in 20% serum. IgG appeared to act like a modulator of factor H and I because it did not stimulate inactivation of C3b-containing complexes in factor I-deficient serum. Thus, the known partial protection of C3bn-IgG complexes from inactivation by factor H and I was downregulated by high concentrations of IgG. The ability of high doses of IgG to stimulate complement inactivation is a novel regulatory role of IgG. This may be one of the molecular principles for its therapeutic efficacy in treating complement-mediated inflammations.

scholarly journals Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- and lipid-binding properties of Tau

2020 ◽  
Vol 295 (23) ◽  
pp. 7905-7922 ◽  
Author(s):  
Nadine Ait-Bouziad ◽  
Anass Chiki ◽  
Galina Limorenko ◽  
Shifeng Xiao ◽  
David Eliezer ◽  
...  
Keyword(s):  
Lipid Binding ◽  
Binding Properties ◽  
Post Translational Modifications ◽  
Unique Role ◽  
Tyrosine Residues ◽  
Normal Functions ◽  
Tau Aggregation ◽  
Β Sheet

The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau's normal functions and its pathogenic properties in tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. Here, we sought to determine the relative contributions of phosphorylation of one or several of the five tyrosine residues in Tau (Tyr-18, -29, -197, -310, and -394) to the regulation of its biophysical, aggregation, and functional properties. We used a combination of site-specific mutagenesis and in vitro phosphorylation by c-Abl kinase to generate Tau species phosphorylated at all five tyrosine residues, all tyrosine residues except Tyr-310 or Tyr-394 (pTau-Y310F and pTau-Y394F, respectively) and Tau phosphorylated only at Tyr-310 or Tyr-394 (4F/pTyr-310 or 4F/pTyr-394). We observed that phosphorylation of all five tyrosine residues, multiple N-terminal tyrosine residues (Tyr-18, -29, and -197), or specific phosphorylation only at residue Tyr-310 abolishes Tau aggregation and inhibits its microtubule- and lipid-binding properties. NMR experiments indicated that these effects are mediated by a local decrease in β-sheet propensity of Tau's PHF6 domain. Our findings underscore Tyr-310 phosphorylation has a unique role in the regulation of Tau aggregation, microtubule, and lipid interactions. These results also highlight the importance of conducting further studies to elucidate the role of Tyr-310 in the regulation of Tau's normal functions and pathogenic properties.

scholarly journals Revisiting the Role of Glycosylation in the Structure of Human IgG Fc

2012 ◽  
Vol 7 (9) ◽  
pp. 1596-1602 ◽  
Author(s):  
M. Jack Borrok ◽  
Sang Taek Jung ◽  
Tae Hyun Kang ◽  
Arthur F. Monzingo ◽  
George Georgiou
Keyword(s):  
Human Igg

Predominant Complement Mediated Lysis of B-CLL Cells by Therapeutic MAbs Rituximab and Campath-1H in Whole Blood Assays.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3445-3445 ◽  
Author(s):  
Josee Golay ◽  
Luca Bologna ◽  
Elisa Gotti ◽  
Alessandro Rambaldi ◽  
Renato Bassan ◽  
...  
Keyword(s):  
Target Cell ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Human Igg ◽  
Blood Samples ◽  
Expression Levels ◽  
Cd20 Expression

Abstract Abstract 3445 Poster Board III-333 The mechanism of action of unconjugated MAbs such as Rituximab and Campath-1H in vivo is still a matter of debate. Most in vitro assays with antibodies rely upon purified effector cells or proteins taken outside their natural context, and on target cell lines rather than patients cells. In order to analyse the activity of therapeutic MAbs on circulating leukemic cells in more physiological conditions and in a system the least manipulated as possible, we have set up a whole blood assays using Rituximab and Campath-1H. Peripheral blood samples were drawn from B-CLL patients or normal donors in sodium citrate and antibodies were directly added at different concentrations. We first demonstrated that neither apoptosis, induced by cross-linked anti-CD20 antibody, nor complement mediated cytotoxicity (CDC) induced by Campath-1H or Rituximab were significantly inhibited by citrate used at the standard concentration (0.1 M). We then performed a number of experiments using whole blood samples in citrate, into which increasing concentrations of Rituximab or Campath-1H were added. Lysis was analysed by FACS analysis after different incubation times at 37°C. We observed that Campath-1H very rapidly and efficiently lysed normal B cells or B-CLL targets in vitro in whole blood: maximal lysis was reached within 4 hours and was observed already with 1 and 10 μg/ml antibody (61 %), even though it was still more effective at 25 or 50 μg/ml (up to 90 % lysis). 25 μg/ml is known to be reached in the circulation after 30mg infusions of the antibody 3 times a week. Lysis by Campath-1H was fully complement dependent since it was inhibited by 90% in presence of excess blocking anti-C5 antibody Eculizumab (200 μg/ml). Eculizumab alone in contrast had no effect on cell viability. We then analysed the efficacy of increasing concentrations of Rituximab in the same assay conditions. We observed in general a much reduced lysis with Rituximab compared to Campath-1H, even using antibody up to 200 μg/ml, a concentration that is reached in the circulation after standard 375 mg/m2 administration of the antibody once a week. Lysis showed also slower kinetics, with limited lysis at 4 hours (mean 6.4%) and maximal lysis with Rituximab reached only after 24 hours incubation (mean 18.8%). Also in this case, target cell death was inhibited by at least 90% in presence of Eculizumab, suggesting a major role of complement. Lysis by Rituximab correlated directly with CD20 expression levels (R=0.8) in 13 B-CLL samples analysed, as expected for a mechanism complement dependent. Indeed a mean 29.3% and 73.2% killing could be observed in the two CD20 bright B-CLL, at 4 and 24 hours respectively, whereas a mean of 3.1% and 10.9% lysis was observed in the 11 low-intermediate CD20 samples analysed at the same time points. These data in whole blood confirm our previously published results on the role of CD20 expression levels in CDC of isolated B-CLL cells (Golay et al., Blood 98, 3383-3389, 2001). In contrast to CDC and apoptosis, ADCC was strongly inhibited by citrate as well as several anti-coagulants tested and therefore could not be analysed in this type of assay. Nonetheless in B-CLL samples, NK cells were below detection limit (<0.1%) in most cases analysed, suggesting that ADCC in the circulation is not a major mechanism of lysis in this disease subtype. Finally we determined the effect of citrate on phagocytosis mediated by Rituximab and in vitro differentiated human macrophages. Phagocytosis could be observed in presence of 0.1M citrate (31%, compared to 44% in absence of citrate). Phagocytosis of B-CLL in whole blood was therefore analysed by layering samples directly onto the macrophages. We observed that phagocytosis of B-CLL targets in whole blood was very low (less than 1% over background) compared to a mean of 47% for purified B-CLL targets phagocytosed in normal culture medium. Phagocytosis in whole blood was low presumably due to the presence of high concentration of human IgG in whole blood since as low as 50 μg/ml human IgG is known to inhibit phagocytosis by 90%. We conclude that the major activity of Campath-1H and Rituximab in the circulation is through complement. Apoptosis, ADCC and phagocytosis appear to play a marginal role in this context but may become more important in tissues. The method presented could be used to rapidly screen novel antibodies for their efficacy through either as apoptosis or CDC directly on unmanipulated patients material. Disclosures No relevant conflicts of interest to declare.

Beclin-1 Phosphorylation By BCR-ABL Is Crucial for CML Leukemogenesis By Suppression of Autophagy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 16-16 ◽  
Author(s):  
Chuanjiang Yu ◽  
Sivahari Prasad Gorantla ◽  
Tony Mueller ◽  
Lena Lippert ◽  
Zhenyu Yue ◽  
...  
Keyword(s):  
Overall Survival ◽  
Specific Substrate ◽  
Autophagosome Formation ◽  
Tyrosine Residues ◽  
Abl Kinase ◽  
Autophagy Induction ◽  
Cml Model ◽  
Tki Treatment ◽  
The Impact

Abstract The constitutively activated chimeric Tyrosine kinase BCR-ABL is critical for initiation, progression and maintenance of chronic myelogenous leukemia (CML). Imatinib and second generation BCR-ABL tyrosine kinase inhibitors (TKIs) serve now as standard therapies for Ph+-patients. However, disease persistence occurs frequently and insensitivity of CML stem cells to TKI treatment is discussed as one major reason for this. Recent evidence accumulates, that autophagy, a genetically-regulated process of adaptation to metabolic stress, is involved in TKI-induced cell death. It is hypothesized, that TKI-induced autophagy could allow CML stem cells to become metabolically dormant enabling their survival under conditions that may mimic growth factor deprivation and thereby "antagonize" TKI-induced cell death. However, the molecular mechanism of BCR-ABL and TKI induced autophagy as well as its role as tumor suppressor or promoter is poorly understood. In our study, we aim to identify the precise role of autophagy and its´ effector molecules in a murine CML model. To test whether BCR-ABL regulates autophagy, we measured LC3 as a marker for autophagy in BCR-ABL+-K562 cell. Interestingly, inhibition of BCR-ABL activity by nilotinib led to increased LC3-II expression and punctual LC3 accumulation, indicating, that BCR-ABL activity can suppress autophagy. Consistent with this, Ba/F3 cells expressing BCR-ABL WT induce autophagy, whereas Ba/F3 cell expressing BCR-ABL-T315I fail to induce autophagy by nilotinib treatment, pointing to a BCR-ABL specific autophagy induction than an unspecific effect of TKI treatment. Next, we investigated the proteins involved in BCR-ABL mediated autophagosome formation. Recruitment of VPS34 and ATG14 to Beclin1 was increased in case of nilotinib treatment and could thereby positively regulate autophagosome formation, whereas Rubicon, a negative regulator was less recruited to the Beclin1-complex. To further identify the impact of Beclin1 as a key regulator of autophagy in BCR-ABL-driven leukemia, we used a targeted genetic approach in a CML mouse model. Interestingly, mice transplanted with Belin1 knockdown, BCR-ABL expressing bone marrow showed a less aggressive disease with significantly lower WBC-count, leukemic burden and prolonged overall survival of the mice. In contrast, deletion of ATG5, another central regulator of autophagy, was not able to change disease onset or progression in the CML model. To further clarify the function of Beclin1, we performed biochemical binding analyses and were able to show, that Beclin1 binds to BCR-ABL independent of BCR-ABL kinase activity and Beclin1 is phosphorylated by BCR-ABL. Interestingly, Beclin1 is an exclusive target of BCR-ABL and can not be phosphorylated by other aberrantly activated tyrosine kinases like Flt3-ITD, NPM-ALK and PDGFRA-D842V. In vitro kinase assay with active ABL-kinase confirm Beclin1 as a specific substrate of BCR-ABL. GST pulldown experiments mapped the N-terminal region of Beclin1 to interact with BCR-ABL. Cloning of different phospho-deficient mutants identified tyrosine residues Y233 and Y352 of Beclin1 as the crucial sites for specific BCR-ABL phosphorylation. To test the impact of BCR-ABL mediated Beclin1-phosphorylation on autophagy induction, we generated Beclin1 phospho-mimic (Y233E/Y352E) and phospho-deficient (Y233F/Y352F) mutations. Interestingly, nilotinib treatment fails to induce autophagy in cells expressing the Beclin1 phospho-mimic mutations, thereby highlighting the necessity of Beclin1 in BCR-ABL-mediated autophagy. Expression of Beclin1 mutations in Beclin1 knockout MEFs and K562 cells show decreased binding of UVRAG, ATG14 and VPS34 to Beclin1 Y233E/Y352E, suggesting an important role of Beclin1 phosphorylation for complex stabilization and autophagy suppression. Taken together our findings identify Beclin1 as a specific substrate of BCR-ABL. Downregulation of Beclin1 is associated with a prolonged overall survival of BCR-ABL transplanted animals; direct phosphorylation of Beclin1 on Tyrosine residues Y233 and Y352 lead to LC3 inhibition and suppression of autophagy. Our results thereby highlight the importance of Beclin1 in BCR-ABL-mediated leukemogenesis and show, that autophagy induction in CML cells may be rather a specific Beclin1-BCR-ABL interaction effect than a general microenvironmental stress phenomenon. Disclosures No relevant conflicts of interest to declare.

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